Identification And Functional Analysis Of Srnas Whose Expression Is Affected By ZnSO₄and EDTA In Xanthomonas Campestris Pathovar Campestris

Bacterial small RNAs (sRNAs) are RNA molecules in the size of50to500nucleotides (nt). Although sRNAs do not contain open reading frame (ORF) and thus do not encode protein but have an independent biological function. It has been known that sRANs are widely spread in both Gram-positive and Gram-negative bacteria, and play an important regulatory role in various bacterial cellular processess including carbon metabolism, cell movement, quorum sensing, virulence, as well as stress response. It is well known that bacteria is able to adapt to differnet environmental stress, and recent studies shown that sRNAs play an important role in the adaptation of bacteria to environmental stresses. Among numerous environmental stresses, heavy metal stress (excess or deficient of heavy metal ion) is one of the most important environmental stresse that bacteria usually encountered under natural condition. Although it has been demonstrated that sRNAs play a key role in the regulation of iron metabolism, nothing is known about the role of sRNAs in the metabolism of other heavy metal ions including zinc。Xanthomonas campestris pathovar campestris(Xcc) is the pathogen of the black rot of cruciferous crops such as cabbage, radish, cabbage and rape, and is a model bacterium for studying the molecular mechanism of pathogenesis of pathogenic bacteria. To identify the sRNA that is involved in the tolerance of Xcc to high zinc and zinc deficiency conditions, in this study, the sRNAs whose expression is affected by ZnSO4and EDTA were identified by the deep RNA sequencing (RNA-seq), and then the biological function of these sRNAs are investigated.For genome wide identification of the sRNAs whose expression is affected by ZnSO4and EDTA, total RNAs were extracted from Xcc strain8004(Xcc8004) grown to exponential phase in the rich medium NYG, NYG supplement with ZnSO4(to the final concentration of350μM), or NYG supplement with EDTA (to the final concentration of300μM), respectively. After remove rRNA, the total RNAs were separated by polyacrylamide gel electrophoresis and RNA molecules in the size of50-200nt and201-500nt were recovered, and then the Solexa cDNA library was constructed and the library was sequenced by the Illumina HiseqTM2000sequencing platform. Finally, the Solexa sequencing data were analyzed by using the method established in our lab previously, and the sRNA candidates whose expression is affected by ZnSO4and EDTA were identified and further confirmed by Northern Blotting or semi-quantitative RT-PCR. Analysis of the Solexa sequencing data, by using a5-fold difference (≥5-fold) cutoff, revealed that the expression of222sRNAs is affected by ZnSO4(81are induced and145are suppressed), and540affected by EDTA (334are induced and206are suppressed). Among these762RNAs,108sRNAs are affected by both ZnSO4and EDTA (47are induced and61are suppressed).17sRNA candidates were selected randomly to detect their expression level in cells growing in NYG, NYG with ZnSO4and NYG with EDTA by quantitative Northern blotting or semi-quantitative RT-PCR. We found that the Northern blotting or semi-quantitative RT-PCR results are well fit with the Solexa sequencing data, suggesting that our Solexa sequencing analysis is reliable at both the qualitative and quantitative level.Two methods, Northern blotting and strand-specific RT-PCR, were used to determine the transcriptional direction of the identified sRNAs. Up to date, the transcriptional direction of11sRNAs has been determined.To determine the role the sRNAs whose expression is affected by ZnSO4and EDTA in Xcc heavy metal stress response, deletion mutants and over-expression strains of these sRNAs were constructed. Until now, we have finished the constructions of6sRNAs’s deletion mutants and over-expression strains. The results of EDTA and heavy metal ion (including Zn2+, Ni2+, Co2+, Mn2+, Cd2+and Cu2+) tolerance assay show that, there was no significant difference in the ability to tolerance to EDTA and the heavy metal ion tested among the deletion mutants, the over-expression strains, and the wild type strain Xcc8004. This suggests that the6sRNAs are not involved or just play a minor role in Xcc response to high zinc concentration and heavy metal ion deficiency. Furthermore, the virulence, virulence factor production and cell mobility of the sRNA deletion mutants and over-expression strains were tested, and the results show that, there was no obvious different compared to that of wild type strain Xcc8004, suggesting that these sRNAs were not involved in or just play a minor role in the cellular processes related to the tested phenotypes。In summary, in this work, by using RNA-seq, several hundred of Xcc sRNAs whose expression is affected by ZnSO4and EDTA were identified, and17of which were further confirmed by quantitative Northern blotting or semi-quantitative RT-PCR。Although no sRNA is found playing key role in Xcc heavy metal stress response, our study provide the first step towards study of sRNAs involved heavy metal stress response of Xcc.